Engineering of sturdy and tolerant process organisms has become an attractive goal for bioprocessing, including biorefinery. Typical bioprocess organisms, such as model organisms Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae), are commonly used because of their extensive characterization as well as the availability of genetic toolboxes enabling easy genetic manipulation. Desirable phenotypes can be found in less characterized or unknown microorganisms (e.g., metagenomics), and will be beneficial if implemented or transferred into the model organisms to improve their bioprocessing characteristics. The desirable phenotype may be due to a known or unknown genotype.
Where the underlying genetics for a desirable phenotype are known, traditional metabolic engineering approaches can be used to transfer desirable traits from an organism to a model or production organism (e.g., E. coli.). As a result, expression of genes necessary for a desirable phenotype can be effectively achieved by hijacking the transcription machinery of a host organism by, for example, using host promoters as well as optimized ribosomal binding sites.
Where the underlying genotype for a desirable phenotype is not known and the desirable phenotype (e.g., tolerance) is complex, meaning that multiple genetic programs are involved, an effective screening mechanism needs to be employed first to elucidate the underlying genetics of the desired phenotype (e.g., screening of genomic libraries). It would be beneficial to employ the screening mechanism in a host organism for the implementation of the desired phenotype because this enables the finding of only important and functional parts of a potential wide span of genetic programs involved in the desired phenotype.
The basis for such a screening mechanism is generally functional expression of foreign genes in a host organism. In the circumstances of complex phenotypes, such a screening mechanism also enables identification of beneficial interactions among the foreign genes. Therefore, another limitation to be overcome is the number of foreign genes that can be screened in single cells, where they can potentially interact. Common genetic elements employed for transfer of large heterologous DNA fragments into a host organism include fosmids and bacterial artificial chromosome's (BACs). Unfortunately, large heterologous DNA fragments in these genetic elements cannot be effectively employed to hijack the host transcription system by engineering strong promoters in front of the heterologous DNA fragments. The first transcriptional stop signal present in the heterologous DNA would lead to an end of transcription leaving all the genetic information located in genes behind this first stop signal un-transcribed. So, such genetic information would not be available for screening and, therefore, negate the benefits of having large genetic elements (e.g., BACs). The combinatorial space of interactions between the genes located in the large genetic elements (e.g., BACs) can only be exploited if the genes are expressed.
To date, there remains a need for a method to screen for microorganisms exhibiting a complex phenotype, which underlying genotype is not known.